Recombinant poxvirus for chimeric proteins of the human immunodeficiency virus

ABSTRACT

The invention relates to HIV chimeric gene formed by the union of fragments of different genes of said virus, wherein said fragments contains epitopes for cytotoxic T cells (CTL) or HIV-1 auxiliary T cells, which are presented by a wide range of antigens of type Major Histocompatibility Complex (HLA-I). Recombinant poxviruses are obtained from said genes, which are useful for prophylactic and therapeutic vaccination against HIV/AIDS infections, are capable of generating a protective immune cell response in vaccinated laboratory animals and are recognized by the CTL lymphocytes of HIV/AIDS patients.

FIELD OF THE INVENTION

[0001] The present invention is related to the field of immunology andin particular with the development of vaccines for the prevention ortreatment of Acquired Immunodeficiency Syndrome (AIDS). Chimeric genesand Fowlpox Viruses expressing thereof, useful for the treatment andprevention of AIDS are disclosed.

[0002] Previous Technique

[0003] HIV is the etiological agent of AIDS (Popovic M, Sarngadharan M,Read G, and Gallo R C. Science 1984, 224:497-500). This virus infectsnot only CD4+ T cells (Klatzman D, Barre Sinoussi F, Nugeyre M T,Dauguet C, Vilmer E, Griscelli C, Brun-Vezinet F, Rouzioux C, Gluckman,J D, Chermannn J C and Montagnier L. Science 1984, 225:59-63) but alsoother cell types such as macrophages, dendritic cells, microglia andepithelial cells.

[0004] HIV can escape from the host immune response in spite of the highlevels of antibodies that persists through all the infection. At thelong term, HIV causes profound immunodeficiency in the host, whichbecomes highly susceptible to the attack of opportunistic infections.

[0005] More than 36 millions persons are living with HIV/AIDS and 94% ofthe 16 000 daily infections occur in developing countries. (UNAIDS.Report on the global HIV/AIDS epidemic, June 2000). Due to thesealarming figures and the absence of an effective and affordabletreatment for this disease, there is an urgent need for the developmentof an HIV vaccine.

[0006] Among several characteristics of HIV that difficult this task themore important is perhaps the high degree of genetic variability of itsantigens, especially the envelope glycoproteins (gp160) where the maindomains involved in the infectious process and targeted by neutralizingantibodies are located.

[0007] Vaccine candidates based on neutralizing antibodies have beenable to protect against HIV in chimpanzees (Berman P W, Gregory T J,Lavon R, Nakamura G R, Champe M A, Porter J P, Wurm F M, Hershberg R D,Cobb G K and Eichberg J W. Nature 1990, 345: 622-625; Girard M, Kieny MP, Pinter A; Barre-Sinoussi F, Nara P, Kolbe H, Kusumi K, Chaput A,Rainhart T, Muchmore E, Ronco J, Kaczorek M, Gomard E, Gluckman J C andFultz P N, PNAS 1991, 88: 542-546). However those experiments wereperformed in nearly ideal conditions where the dose, route and timing ofthe viral challenge were very different from natural infection.Moreover, those immunogens can't protect against divergent HIV isolatesand the antibodies raised fail to neutralize primary HIV isolates.

[0008] Different vaccine candidates have been evaluated in Phase I andII clinical trials (Johnston M I. AIDS vaccine development: status andfuture directions. 1999. XII Colloque des Cent Gardes. Ed. Girard M andDodet B.161-163). Most of these are based on the envelope proteins:gp160 and gp120. Only one vaccine, based on recombinant gp120 iscurrently undergoing efficacy evaluation in Phase III trials in Thailandand USA. Results from previous trials suggested that only very limitedprotection if any can be expected from this vaccine.

[0009] Due to these serious limitations to generate a humoral responseable to confer protection against different HIV isolates and subtypes,the efforts of the investigators have mostly switched in the last yearstoward the development of vaccine candidates capable of stimulate mainlythe cellular branch of the immune system and particularly cytotoxic Tcells directed against HIV antigens.

[0010] Among the experimental findings that strongly suggest theclinical relevance of anti HIV CTIs are: The administration of anti CD8monoclonal antibody to macaques previously inoculated with Simian-HumanImmunodeficiency Virus (SHIV) markedly enhanced the levels of viremia(Matano T, Shibata R, Simeón C, Connors M, Lane C, Martin M,Administration of an Anti-CD8 monoclonal antibody interferes with theclearance of chimeric Simian/Human Immunodeficiency virus during primaryinfections of rhesus macaques, J Virol, 1998, 72, 1: 164-169); viralvariants able to escape CD8+ T cell recognition are selected in bothHIV-infected individuals (Borrow, P., H. Lewicki, X. Wei, M. S. Horwitz,N. Peffer, H. Meyers, J. A. Nelson, J. E. Gairin, B. H. Hahn, M. B. A.Oldstone, and G. M. Shaw. 1997. Antiviral pressure exerted byHIV-1specific cytotoxic T lymphocytes (CTLs) during primary infectiondemonstrated by rapid selection of CTL escape virus. Nature Med.3:205-211.) and SIV-infected macaques (Allen, T. M., O. C. D H, P. Jing,J. L. Dzuris, B. R. Mothe, T. U. Vogel, E. Dunphy, M. E. Liebl, C.Emerson, N. Wilson, K. J. Kunstman, X. Wang, D. B. Allison, A. L.Hughes, R. C. Desrosiers, J. D. Altman, S. M. Wolinsky, A. Sette, and D.I. Watkins. 2000. Tat-specific cytotoxic T lymphocytes select for SIVescape variants during resolution of primary viraemia. Nature.407:386-90); SCID mice populated with PBMC from volunteers injected withHIV-1 recombinant Vaccinia Virus (VV) were protected against challengedin the absence of neutralizing antibodies (Van Kuyk R, Torbett B,Gulizia R et al, Human CTL specific for the nef protein of HIV protecthu-PBL-SCID mice from HIV infection. AIDS Res Hum Retroviruses, 1993; 9(suppl 1:S77); a significant proportion of exposed uninfected personsdisplay cellular immune response specific for. HIV proteins, this istrue for African sex workers (Rowland-Jones S L, J Sotton, K Ariyoshi, TDong, F Gotch, s McAdams, D Whitby, S Sabally, A Gallimore, T Corrah, MTakiguchi, T Schltz, A McMichael, H Whittle. 1995. HIV-specificcytotoxic T cells in HIV-exposed but uninfected Gambian women. NatureMedicine, 1: 59-64) and children born from seropositive mothers(Rowland-Jones S L, D F Nixon, M C Aldhous, F Gotch, K Aroyoshi, NHallam, J S Kroll, K Froebel, A McMichael. HIV specific cytotoxic T-cellactivity in an HIV-exposed but uninfected infant. Lancet, 1993, 341:860-861). Additionally long term non progressors exhibit a strong CTLresponse (Cao, Y, Qin L, Zhang I, Safrit J and Ho D D, New Engl J Med,1995, 332:201-208; Riviere Y, McChesney M B, Porrot E, et al. AIDS ResHum Retroviruses, 11:903-990); and the HLA class I type has beenassociated with the rate of disease progression in HIV-1-infectedindividuals (Carrington, M., G. W. Nelson, M. P. Martin, T. Kissner, D.Vlahov, J. J. Goedert, R. Kaslow, S. Buchbinder, K. Hoots, and O. B. SJ. 1999. HLA and HIV-1: heterozygote advantage and B*35-Cw*04disadvantage. Science. 283:1748-52). The CTL response precede theneutralizing antibodies in the natural infection and has been associatedwith the control of viremia in acute infection (Koup R A, Safrit J T,Cao Y, et al, Temporal association of cellular immune responses with theinitial control of viremia in primary human immunodeficiency syndrome, JVirol, 1994; 68: 4650-4655) and progression to AIDS correlates stronglywith the impairment of CTL activity. (Harrer T, Harrer E, Kalams S,Elbeik T, Staprans S, Feinberg M B, Cao Y, Ho D D, Yilma T, Caliendo A,Jonson R P, Buchbinder S, and Walker B. HIV-specific CTL-response inhealthy long-term asymptomatic HIV infection. AIDS Res Hum Retroviruses,1996, 12, 7: 585-592). Finally vaccines that induce virus-specific CD8+T cell responses can favorably affect the outcome of infection in SIVmodels of HIV infection (Barouch, D. H., S. Santra, J. E. Schmitz, M. J.Kuroda, T. M. Fu, W. Wagner, M. Bilska, A. Craiu, X. X. Zheng, G. R.Krivulka, K. Beaudry, M. A. Lifton, C. E. Nickerson, W. L. Trigona, K.Punt, D. C. Freed, L. Guan, S. Dubey, D. Casimiro, A. Simon, M. E.Davies, M. Chastain, T. B. Strom, R. S. Gelman, D. C. Montefiori, M. G.Lewis, E. A. Emini, J. W. Shiver, and N. L. Letvin. 2000. Control ofviremia and prevention of clinical AIDS in rhesus monkeys bycytokine-augmented DNA vaccination. Science. 290:486-92; Gallimore, A.,M. Cranage, N. Cook, N. Almond, J. Bootman, E. Rud, P. Silvera, M.Dennis, T. Corcoran, J. Stott, A. McMichael, and F. Gotch. 1995. Earlysuppression of SIV replication by CD8+ nef-specific cytotoxic T cells invaccinated macaques. Nature Med. 1:1167-1173.)

[0011] All this body of experimental findings strongly suggest thattherapeutic and prophylactic strategies should include theinduction/preservation/restoration of this arm of the immune response asat least one of their goals.

[0012] Different methodologies have been developed to generate CTLs inanimals or humans. The most effective so far has been the recombinantlive vectors. This method uses harmless viruses or bacteria to transportselected genes from the pathogen into the cells of the recipient toproduce there the selected antigens. This procedure of gene deliveringinto cells maximizes the processing of CTL epitopes and theirpresentation by MHC-I molecules and subsequently the efficientstimulation of CTL clones in the host.

[0013] The viruses that have been more successfully used as vectors havebeen the poxviruses (Poxyiridae family). The best-known member of thisfamily is Vaccinia Virus (VV), which was extensively used in humansduring smallpox eradication campaign.

[0014] Several clinical trials has been carried out with VV recombinantfor HIV proteins (Corey L, McElrath J, Weihold K, Matthewa T, StableinD, Grahm B, Keefer M, Schwartz D, Gorse G. Cytotoxic T Cell andNeutralizing Antibody Responses to Human Immunodeficiency Virus Type 1Envelope with a combination vaccine regimen. J Infectious Dis, 1998,177:301-9; Graham B S, Matthews T J, Belshe R, Clements M L, Dolin R,Wright P F, Gorse G J, Schwartz D H, Keefer M C, Bolognesi D P, Corey L,Stablein D, Esterlitz J R, Hu S L, Smith G E, Fast P, Koff W, JInfectious Dis, 1993, 167: 533-7). However, VV has two main limitationsfor human use: (1) A small percentage of vaccinated persons showedstrong adverse reactions that can be lethal in the case ofimmune-compromised individuals (2) persons with previous history of VVvaccination respond poorly against heterologous antigens.

[0015] A solution to these drawbacks has been the use of Avipoxvirusinstead of VV. These are members of the poxvirus family but theirreplication is restricted to avian cells and its replication cycle isabortive in human cells. Two Avipoxviruses have been used with thesepurposes: Canarypox Virus (CPV) and Fowlpox Virus (FPV).

[0016] Avipoxviruses recombinants for various human pathogens oftumor-associated antigens induce CTL response in animals (Limbach K J,and E Paoletti. 1996. Non-replicating expression vectors: applicationsin vaccines development and gene therapy. Epidemiol. Infect.116:241-256). The use of recombinant Avipoxvirus for vaccine developmenthas been patented in USA (Paoletti E. y cols 1992 U.S. Pat. No.5,174,993, Paoletti E. et al 1993, U.S. Pat. No. 5,505,941) andspecifically a patent application on the use of recombinantavipoxviruses for lentiviral antigens has been presented in Europe.(Paoletti E et al, EP0956360)

[0017] A CPV recombinant for HIV-1 gag, pol and env has been evaluatedin Phase 1 and 11 trials in healthy volunteers (Clements-Mann M L, KWeinhold, T J Matthews, B S Graham, G L Gorse, M C Keefer, M J McElrath,R-H Hsieh, J Mestecky, S Zolla-Pazner, J Mascola, D Schwartz, RSiliciano, L Corey, P F Wright, R Belshe, R Dolin, S Jackson, S Xu, PFast, M C Walker, D Stablein, J-L Excler, J Tartaglia, A-M Duliege, FSinangil, E Paoletti. 1998. Immune responses to Human ImmunodeficiencyVirus (HIV) Type 1 induced by Canarypox expressing HIV-1MN gp120,HIV-1SF2 recombinant gp120, or both vaccines in seronegative adults. JInfect Dis 177: 1230-1246; Egan M A, W A Pavlat, J Tartaglia, EPaoletti, K J Weinhold, M L Clements, R F Siliciano. 1995. Induction ofHuman Immunodeficiency Virus Type 1 (HIV-1)-specific cytolytic Tlymphocyte responses in seronegative adults by a nonreplicating,host-range-restricted canarypox vector (ALVAC) carrying the HIV-1MN envgene. J Infect Dis 171: 1623-1627). CTLs against at least one HIVantigen were reported in the 50% of vaccinated in a Phase I trial, 30%in a Phase II trial and less than 10% in the last Phase I trial inUganda. This rCPV (vCP205) was created trough the insertion of HIV genesin three different non-essential regions in the genome to achieve a CTLresponse against more than one HIV target.

[0018] In the other hand FPV has been also used to induce a CTL responsein macaques against HIV antigens in combination with DNA immunization.(Robinson H L, D C Montefiori, R P Johnson, K H Manson, M L Kalish, J DLifson, T A Rizvi, S Lu, S-L Hu, G P Mazzara, D L Panicali, J G Herndon,R Glickmanm, M A Candido, S L Lydy, M S Wyand and H M McClure. 1999.Nature Medicine, 5: 526-534). This combination of immunogens providedsome level of protection in the HIV-1/macaca nemestrina infection model(Kent S J, A Zhao, S J Best, J D Chandler, D B Boyle, I A Ramshaw.Enhanced T-Cell immunogenicity and protective efficacy of a humanimmunodeficiency virus type 1 vaccine regime consisting of a consecutivepriming with DNA and boosting with recombinant fowlpox virus. 1998. JVirol, 72: 10180-10188). However this animal model present importantlimitations since HIV infection in M nemestrina is inefficient anddifficult to reproduce.

[0019] It has also been reported the generation of a CTL responsethrough the immunization with minigenes composed of a series of exactCTL epitopes from several pathogens (Whitton, L, Sheng N, Oldstone M B,and McKee T. A “string of beads” vaccine, comprising linked minigenes,confers protection from lethal-dose virus challenge, J Virol, 1993, 67,1:348-352; A multivalent minigene vaccine, containing B-cell, cytotoxicT-Lymphocyte and Th epitopes from several microbes, induces appropriateresponses in vivo and confers protection against more than one pathogen.J Virol, 71, 3: 2292-2302).

[0020] Modified Vaccinia Ankara (MVA) recombinant for a gag derivedminigene together with the whole gag gene has been used to induce a CTLresponse in mice (Hanke T, R V Samuel, T J Blanchard, V C Neumann, T MAllen, J E Boyson, S A Sharpe, N Cook, G L Smith, D I Watkins, M PCranage, A J McMichael. 1999. Effective induction of simianimmunodeficiency virus-specific cytotoxic T lymphocytes in macaques byusing a multiepitope gene and DNA prime-Modified Vaccinia Virus Ankaraboost vaccination regimen. J Virol, 73, 9: 7524-7532). Those minigenesconsist of a string of discrete CTL epitopes from gag.

[0021] The main limitation of the minigene approach is that thecombination of individual CTL epitopes only covers a limited range ofHLA antigens and therefore the CTL response elicited is by definition tomuch restricted.

DESCRIPTION OF THE INVENTION

[0022] The essence of the present invention is the construction ofchimeric genes composed by CTL epitopes rich regions from HIV proteins,where those regions are selected from both, internal conserved proteinsand regulatory proteins expressed very early in the viral life cycle.

[0023] This solution has advantages over the described HIV minigenesbecause allows the simultaneous processing of overlapping CTL epitopespresented by many HLA alleles. Another advantage of this solution incomparison to other avipoxvirus recombinant for several HIV-1 proteinsis that the concentration of immunologically relevant regions fromseveral proteins in a single gene facilitates the generation ofrecombinant viruses, and avoid the necessity to use several antibioticresistance systems in the same recombinant virus. Additionally itfacilitates the combination of epitopes from several HIV subtypes in asingle recombinante virus. The chosen regions belong to the mostconserved viral proteins and to early expressed regulatory products.Those CTL epitopes rich regions are combined with conserved T helpercells epitopes flanked by two lysines to facilitate their processing bycellular proteases. Finally a B cell epitope, recognized by a monoclonalantibody, is added to facilitate the detection of the polypeptide byimmunochemical techniques.

[0024] The chimeric gene is assembled by joining together different DNAfragments, some of them generated by chemical synthesis and othersamplified by Polimerase Chain Reaction (PCR) using HIV genes astemplates. The DNA fragments are cloned together in an appropriateplasmid vector, sequenced and translated to a poxvirus recombinationvector.

[0025] More particularly, this invention refers to the gene cr3, whichcontains Th cells epitopes from HIV-1 proteins gp120, gp41 and Vpr, theepitope on the V3 loop of gp120 recognized by Mab 2C4 (Duarte C A, PerezL, Vázquez J, Dueñas M, Vilarubia O L, Navea L, Valde's R, Reyes O,Montero M, Ayala M, and Gavilondo J. Epitope mapping, V region DNAsequence, and neutralizing Fab fragments of two monoclonal antibodiesagainst the HIV-1 V3 loop. Immunotechnology 1996, 2:11-20) and CTLepitopes rich regions on proteins RT, Gag and Nef.

[0026] Those chimeric genes are inserted in the genome of a bacterial orviral lived vector (ej poxvirus, herpesvirus, alphavirus, poliovirus,adenovirus, BCG, Salmonella), being this vector preferentially apoxvirus, and still more specifically an avipoxvirus and even morespecifically FPV. Those recombinant live vectors are used to induce aTH1 immune response and cytotoxic T cells against HIV in animals orhumans.

[0027] Even more specifically this invention relates to FPV recombinantfor those chimeric proteins and particularly to the recombinant FPVstrains denominated FPCR3 and FPSCR3gpt, which contains the chimericgene cr3. Once assembled as described above cr3 is cloned in a poxvirusrecombination vector, in particular a FPV recombination vector. In thisparticular case plasmids pEFL29 y pFP67xgpt were used as recombinationvectors. pEFL29 presents homologous regions to the 6 kb BamHI terminalfragment of FPB genome, which flanks the transcriptional unit in whichthe heterologous gene is inserted under the control of VV 7.5K promoter,and contains also the reported gene y lacZ under the control of 4bpromoter of FPV. pFP67xgpt employs open reading frames 6 and 7 from the11.2 kb BamHI region as homologous recombination signals. Those regionsflanks the transcriptional unit in which the heterologous gene is placeunder the synthetic poxviral E/L promoter and it also contains the gptgene which confers resistance to mycophenolic acid which allows theselection of recombinant viruses.

[0028] The resultant plasmids were denominated pFPCR3 y pFPSCR3gptrespectively. Those plasmids are transfected in a primary culture ofChicken Embryo Fibroblasts (CEF) using one of the several transfectiontechniques available in the state of the art. In this particular casethe transfection is carried out using lipofectin (Sigma, USA) in CEFpreviously infected with the FP29 strain of FPV but other methods suchas electroporation and DEAE Dextran, among others, can be used. As aresult of the homologous recombination between plasmid and thecorresponding non-essential regions on the FPV genome recombinantviruses, which expressed B galactosidase, can be recovered in the caseof pFPCR3 or resistant to mycophenolic acid in the case of pFPSCR3gpt.The presence of the selection marker allows the identification ofrecombinant viral plaques and their purification by several passages onCEF. The presence of the heterologous gene on the selected viruses canbe verified by PCR and the expression of the protein can be verified bywestern blot.

[0029] This invention relates also to the use of recombinant FPV,obtained as described, to induce a TH1 immune response with CTL activityin Balb/c mice alone or in combination with a pharmaceutically acceptedformulation selected from those in the state of the art.

[0030] This invention refers also to a therapeutic or preventivecombination of recombinant FPV for the described chimeric genes, andparticularly to FPCR3 and FPSCR3gpt, with immunomodulators or adjuvantsin particular with cytokines such as IL2, IL12, IFNγ, GMSCF, GSCF, amongothers, which stimulates preferentially the TH1 immune response.

[0031] Particularly it refers to combination of viruses FPCR3 orFPSCR3gpt with daily doses of IL2 in a range between 10² y 10⁷ iu inanimals or humans. The daily administration of IL2 to Balb/c micestarting the day of the administration of the FPV or after potentiatesthe cellular immune response against CR3.

[0032] Although it refers particularly to CR3, it is in the essence ofthis invention that CTL rich fragments other than those in CR3 orfragments equivalent to those in CR3 but from other HIV-1 isolates canalso be used. Similarly, although it refers particularly to FP9 strainof FPV, it is in the essence of this invention that other FPV parentalstrains can be used to construct the recombinant viruses, as well asanother avipoxvirus such as CPV, other poxvirus such as VV or MVA orstill other viruses such as herpesvirus, alphavirus, adenovirus,poliovirus or even bacterias such as BCG or Salmonella.

[0033] In another embodiment of the present invention the gene can becloned in a proper plasmid vector for expression in mammalian cells andbe injected into a mammal to induce a TH1 immune response and CTLactivity in combination with a pharmaceutically acceptable carrier.

[0034] In still another embodiment of the invention it is also includeda therapeutic or preventive combination of those recombinant plasmidsdescribed above with immunomodulators or adjuvants such as described orstill others such as liposomes, polysaccharides, lipopeptides, lipids,proteoliposomes or combinations thereof.

[0035] In still another embodiment of this invention those genes can beclones in other plasmids for expression of the recombinant proteins inbacteria, yeast, fungi, insect or mammalian cells, plants or in the milkof transgenic animals. The proteins recovered from these systems couldalso be used to induce a TH1 immune response and CTL activity in animalsor humans when administered in an appropriate expressed in apharmaceutically acceptable carrier.

[0036] In still another embodiment of the invention, therapeutic orpreventive combinations of CR3 protein with immunomodulators oradjuvants such as described above or still others such as liposomes,polysaccharides, lipids, proteoliposomes or other adjuvants availableaccording to the state of the art capable to potentiate the TH1 typeimmune response and CTL activity in animals or humans.

DESCRIPTION OF FIGURES

[0037]FIG. 1. Plasmid pEFL-cr3, for the homologous recombination inFowlpox using the ORF-1 from the BamH1 6 Kb terminal region as insertionsite. The gene cr3 is under the control of VV p7.5K promoter and thereporter gene LacZ under FPV 4b promoter.

[0038]FIG. 2. Plasmid pFP67xgpt, for homologous recombination in FPVusing the DNA region between ORF-6 and ORF-7 from the 11.2 kb BamHIfragment as insertion site. The gene cr3 is placed under the control ofthe synthetic promoter E/L and the gene Ecogpt under the control of W7.5K promoter.

[0039]FIG. 3. (A) PCR and (B) Western blot of three independent cr3recombinant FPVs: (1) FPCR3.1; (2) FPCR3.2; (3) FPCR3.3; (4) FPL29; (5)DNA molecular weight marker.

[0040]FIG. 4. (A) PCR with cr3 internal oligonucleotides (B) Westernblot from three independent cr3 recombinant FPV (1) FPSCR3GPT.1; (2)FPSCR3GPT.2; (3) FPSCR3GPT.3; (4) parental virus; (5) Molecular weightmarker.

[0041]FIG. 5. Stability of CR3 expression assessed by Western blot.Lanes represent three independent samples of FPV infected with FPSCR3GPTfrom the viral stock (1,2,3) or purified by sucrose cushion (4,5,6).Lane 7 represents CEF infected with the parental virus.

[0042]FIG. 6. Results from two independent ELISPOT experiments usingsplenocytes from mice immunized with FPSCR3gpt and P815 cells loadedwith peptide 32 or infected with VV recombinant for CR3, Gag or Nef. Theresults are expressed as number of IFN gamma secreting cells per 106splenocytes. The values of the corresponding negative controls (P815alone or VV WR infected) have been subtracted.

[0043]FIG. 7. IFN gamma ELISPOT experiments using splenocytes from miceimmunized with FPCR3 or FPSCR3gpt and P815 stably transfected with thecr3 gene. The results are expressed as number of IFN gamma secretingcells per 10⁶ splenocytes. The values of the negative controls (parentalP815) have been subtracted.

[0044]FIG. 8. Recognition of VVCR3 infected autologous B cells by Tlymphocytes from AIDS patients. The results from an IFNγ ELISPOT areexpressed as the number of IFN gamma secreting cells per 10⁶ peripheralblood mononuclear cells.

EXAMPLES Example 1 Obtention of cr3

[0045] cr3 is a chimeric gene assembled by fragments of different HIVgenes. It was assembled on pTAB11 plasmid, which is essentially equal topTAB9 (Gómez C E, Navea L, Lobaina L, Dubed M, Expósito N, Soto A andDuarte CA. he V3 loop based Multi-Epitope Polypeptide TAB9 Adjuvatedwith Montanide ISA720 is Highly Immunogenic in Nonhuman Primates andInduces Neutralizing Antibodies Against Five HIV-1 isolates. Vaccine17:2311-2319, 1999), but has the T1 and T2 T helper cell epitopes fromgp120 at the 5 end extreme of the gene instead of the fragment encodingfor the N-terminal part of the P64K protein. A 186 bp blunt-BamHIsynthetic DNA fragment encoding for the T2 epitope from gp120, the V3epitope of the MN strain, and T helper cell epitopes from gp41 and vpr,was cloned into pTAB11 previously digested EcoRV-BamHI. DNA sequencesencoding for two consecutive lysines were inserted between individualepitopes to facilitate intracellular processing. The resultant plasmidwas named pCR1. A 603 bp fragment encoding for the p66/p51 (RT) protein(pos. 2663-3109 from HIV-1 SF2 provirus) was PCR amplified using theO.2660 and O.2661 primers (table 1). The PCR fragment was extracted fromlow-melting agarose digested BgIII-EcoRI and subcloned into theBgIII-EcoRI cut pCR1 vector to obtain the pCR2 plasmid encoding for CR2protein. Next, a 324 bp fragment, comprising a sequence of the nef gene(pos. 8516-8818 from HIV-1 LAI isolate), was PCR amplified with primersO.2662 and O.2663. Finally, another segment of 267 bp in the gag gene(pos. 1451-1696 from HIV-1 SF2) was amplified using primers O.2664 andO.2665 (table 1). Then, an overlapping PCR was accomplished using 20pmol of primers O.2662 and O.2666 (table 1). Equal amount of each band(0.47 pmol) were mixed in PCR buffer [KCl 50 mM; Tris-HCl 10 mM, (pH8.3), at 25° C.; gelatin 0.001%], MgCl₂ 2.5 mM, dNTP 0.2 mM each and 4 Uof Taq Polymerase, in a volume of 50 μL. To promote the annealing of thebands by the complementary 9 bp ends of O.2663 and O.2664oligonucleotides, the mixture was first heated at 92° C. for 2 min andthen cooled at 50° C. Finally, the temperature was increased to 72° C.during 5 min to extend the annealed segments. Afterward, 10 μL of theabove reaction was added to a mixture of PCR Buffer containing 2.5 mMMgCl₂, 0.2 mM dNTPs, 20 pmol of O.2662 and 20 pmol of O.2666 and 4 UVent pol. in 50 μL as total volume. Standard amplification conditionswere 92° C. for 2 min, followed by 30 cycles of 92° C. for 40 sec, 50°C. for 1 min and 72° C. for 1 min, and a final extension at 72° C. for 5min. Next, the overlapping nef-p24 amplified band was purified fromelectrophoresis in low-melting agarose and digested with XbaI. Finally,the former blunt-XbaI band was cloned into a pCR2 vector previously cutNruI-XbaI to obtain pCR3 plasmid. cr3 encodes therefore for a chimericproteins which includes T helper cells and CTL epitopes from gp120,gp41, vpr, RT, nef and gag presented by a wide range of HLA antigens(table 2). TABLE 1 DNA SEQUENCE OFOLIGONUCLEOTIDES USED IN PCR REACTIONSOligonucleotide Sequence (5′-3′) 0.2660 GAAGATCTGTACAGAAATGGAAAAG 0.2661GGAATTCTCGCGATCCTACATACAAATCATC 0.2662 GACATCACAAGTAGCAATACAGC 0.2663CCCTGCATGTGGCTCAACTGGTACTAGCTTG 0.2664 GTTGAGCCACATGCAGGGCCTATTGCAC0.2665 GCTCTAGATTATTCGGCTCTTAGAGTTTTATAG 0.2666GCTCTAGATTATTCGGCTCTTAGAG

[0046] TABLE 2 T CELL EPITOPES IN CR3 Epit p s HLAI HLAII p24 87-175 87-101 HAGPIAPGQMREPRG A2  91-110 IAPGQMREPRGSDIAGTTST A2, A24, B13,B38 101-120 GSDIAGTTSTLQEQIGWMTN A26, A30, B38 108-117 TSTLQEQIGWB*5701, B*57, B*5801, B57, B58 121-135 NPPIPVGEIYKRWII B8 121-142NPPIPVGEIYKRWIILGLNKIV B8, B27, A33, B35 122-130 PPIPVGEIY B*3501124-138 IPVGEIYKRWIILGL B8 127-135 GEIYKRWII B8 128-136 EIYKRWIIL B8,B*0801 129-138 IYKRWIILGL A*2402 130-148 YKRWIILGLNKTVRMYSPT B271301-139  KRWIILGLN B27 134-143 IILGLNKIVR A33 136-145 LGLNKIVRMY Bw62136-146 LGLNKIVRMYS B62 137-145 GLNKIVRMY B*1501, B62 151-170LDIRQGPKEPRDYVDRFYK ND 162-172 RDYVDRFYKTL (B44, or A26, or B70),B*4402, A*2402 166-174 DRFYKTLRA B*1402, B14 Nef 43-150 68-76 FPVTPQVPLB*3501, B35, B7 68-77 FPVTPQVPLR B7, B*0702 71-79 TPQVPLRPM B*0702 74-81VPLRPMTY B35 73-82 QVPLRPMTYK A3; A11; B35 74-81 VPLRPMTY B35, B*350175-82 PLRPMTYK A*1101 82-91 KAAVDLSHFL Cw8, C*0802 83-94 AAVDLSHFLKEKA11 84-91 AVDLSHFL Bw62 84-92 AVDLSHFLK A11, A*1101 86-94 DLSHFLKEK A3.1 86-100 DLSHFLKEKGGLEGL A2, B35, C4 90-97 FLKEKGGL B8  92-100 KEKGGLEGLB60, B*4001  93-106 EKGGLEGLIHSQRR A1, B8 102-115 HSQRRQDILDLWIY B7103-127 SQRRQDILDLWIYHTQGYFPDWQNY B13 105-114 RRQDILDLWI B*2305 106-115RQDILDLWIY B27 115-125 YHTQGYFPDWQ B17 116-125 HTQGYFPDWQ B57 117-128TQGYFPDWQNYT B17; B37 117-127 TQGYFPDWQNY Bw62, B*1501 120-128 YFPDWQNYTB*3701, B*5701, B15, B37, B57 120-144 YFPDWQNYTPGPGIRYPLTFGWCYK A24126-137 NYTPGPGVRYPLT B7 128-137 TPGPGVRYPLT B*0702, B*4201, B7,B7(*8101) 130-143 GPGVRYPLTFGWCY B*57 132-147 GVRYPLTFGWCYKLVP B18, A1,B8 133-148 VRYPLTFGWCYKLVPV B57 135-143 YPLTFGWCY B*1801, B18, B35, B49136-145 PLTFGWCYKL A*0201, A2 RT 36-192 36-52 EICTEMEKEGKISKIGP ND 42-50EKEGKISKI B*5101, B51  93-101 GIPHPAGLK A3  98-113 AGLKKKKSVTVLDVGD Cw4103-107 KKSVTVLDVGDAYFS Cw4 107-115 TVLDVGDAY B35, B*3501 108-118VLDVGDAYFSV A*0201, A2 113-120 DAYFSVPL B*5101, B24 118-127 VPLDEDFRKYB35, B*3501 126-135 KYTAFTIPSI A2 128-135 TAFTIPSI B51, B*5101 151-159QGWKGSPAI B*5101 153-165 WKGSPAIFQSSMT B27 156-164 SPAIFQSSM B7, B35,B*3501 158-166 AIFQSSMTK A*0301, A*1101, A3, A*6801, A11, A3.1, B*0301175-142 KQNPDIVIY A*3002 177-185 NPDIVIYQY B35, B*3501 181-189 VIYQYMDDLA2, A*0201 181-191 VIYQYMDDLYV A*0201 172-192 FRKQNPDIVIYQYMDDLYVG DR1,2 ó 3, 4, 7 T1-T2 gp120 421-440 KQIINMWQEVGKAMYAPPIE A2 several 436-442KVGKAMY A2 436-443 KVGKAMYA A2 105-117 HEDIISLWNQSLK A2 several 115-123IISLWNQSL A2.1 gp 41 584-594 ERYLKDQQLLG B14; B8; A24 582-593 YLKDQQLLB8, B*0801, A*2402 580-593 ERYLKDQQLL A*2402, B*0801, B8 581-592RYLKDQQL B14, B*1402 Vpr 66-80 QLLFIHFRIGCRHSR ND

[0047] Numbers represent positions relative to the HXB2 amino acidsequence of each viral protein, the viral isolate is within parenthesis;ND, not defined.

Eixample 2 Cloning of cr3 in pFPL29

[0048] In pCR3, the cr3 gene was cloned under the control of pTryp, witha ClaI site on the 5′and the T4 phage gene 32 terminator and a HindIIIsite at 3′. This plasmid was digested ClaI and HindIII, and treated withKlenow I to obtain a cr3 gene with ATG at the 5′ end and translationstop codons at 3′. This DNA fragment was cloned in the poxvirusrecombination vector pEFL29.

[0049] pEFL29 has the BamH1 6 Kb terminal fragment of FPV asnon-essential regions for homologous recombination in the FPV genome.This fragment contains three ORF and the ORF1 is interrupted. Flanked bythese homology regions are the VV p7.5K, promoter, followed by a SmaIsite and the reporter gene lacZ under the control of the late promoter4b of FPV. This plasmid includes also the kanamycin resistance gene anda bacterial origin of replication.

[0050] PEFL29 was SmaI digested, treated with alkaline phosphatase andligated with a ClaI/HindIII digested band containing cr3 gene. Severalclones with cr3 in the right orientation under the p7.5K were selected.E. coli strain DH5α (φ80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17(rK− mK+), supE44, relA1, deoR, Δ(lacZYA-argF)U169) was used forpropagation and selection of recombinant plasmids in LB mediumcontaining kanamicin (25 μg/ml). All genetic manipulations were madeaccording to Sambrook y col (Sambrook J, Fritsh E F, Maniatis T. 1989.Molecular Cloning. A Laboratory Manual. Sec Ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.)

[0051] The DNA sequenced of clone pEFL-cr3 (FIG. 1), was verified usingan automatic sequence processor (Pharmacia). This clone was purifiedusing CsCl gradient and used to transfect chicken embryo fibroblasts(CEF).

Example 3 Cloning cr3 in pFP67xgpt

[0052] cr3 gene was PCR amplified and cloned in pMosblue vector(Amersham, UK). The resultant plasmid was named pTCR3. pTCR3 wasHpaI/BamHI digested and the shorter band containing cr3 was cloned inthe poxvirus vector pFP67xgpt.

[0053] pFP67xgpt has a fragment of the 11.2 Kb BamH1 of FPV genome asnon-essential region for homologous recombination in the FPV genome(Tomley F, Binns M, Campwell J, Boursnell M. Sequence Analysis of an11.2 Kilobase, near-terminal, Bam HI fragment of fowlpox virus, J GenVirol, 1988, 69, 1025-1040). This fragment contains the open readingframe 6 and 7 of this region and the insertion occurs at the intergenicregion. Flanked by these homologous regions are an E/L syntheticpromoter (Carroll M W, Moss B. E. coli B-glucoronidase (GUS) as a markerfor recombinant vaccinia viruses, Biotechniques, 1995, 19, 3: 352-354),and the reporter gene Ecogpt under the control of 7.5K promoter of VV.This plasmid includes also the kanamycin resistance gene and a bacterialorigin of replication.

[0054] Plasmid pFP67xgp was cut Stul/BamHI and ligated with a cr3containing DNA fragment derived from the Hpal/BamHI digestion of pTCR3.Several clones with cr3 in the proper orientation under the E/Lsynthetic promoter were selected (FIG. 1). E. coli strain DH5a(φ80dlacZΔM15, recA1, endA1, gyrA96, thi-1, hsdR17 (rK− mK+), supE44,relA1, deoR, Δ(lacZYA-argF)U169) was used for propagation and selectionof recombinant plasmids in LB medium containing ampicillin (50 μg/ml).All genetic manipulations were made according to Sambrook et al. The DNAsequenced of clone pFP67xgptct was verified using an automatic sequenceprocessor (Pharmacia). This clone was purified using CsCl gradient andused to transfect CEF).

Example 4 Generation of Recombinant FPVs

[0055] The parental FPV used for the generation of recombinants was theattenuated HP-438 strain, which was derived from the pathogenic strainHP-1 by six consecutive passages on CEFs, two further passages onchorioallantoic membranes, and finally 438 passages through CEFs (Mayr Aand K Malicki. 1966. Attenuierung von virulentem Huhnerpockenvirus inZelikulturen und Eigenschaften des attenuierten Virus. Zentralbi.Veterinaermed. Reihe B 13: 1-13). A twice-plaque-purified isolate ofHP438 (FP9) was then passaged six times to constitute a stock. FPVstocks were grown on CEFs in 199 medium containing 2% newborn calf serum(NBCS).

[0056] Recombinant FPV were generated by homologous recombinationbetween FP9 and plasmid pEFL29 or its derivatives as previouslydescribed. CEFs grown in 25 cm2 flasks were infected with FP9 at amultiplicity of infection (m.o.i) of 2 plaque forming units (pfu)/cell,then 2 hours later the cells were transfected with CsCl purified 10 μgof plasmid DNA (pEFL-cr3 or pFP67xgptctl) using 20 μg of Lipofectin(Gibco BRL, USA). Fresh medium (3 ml of 199 medium containing 10%tryptose phosphate broth plus 2% NBCS) was added and the cells wereincubated at 37° C. in a CO₂ incubator. Fresh medium was added againafter 24 h, and then the cells were incubated for a further 3 to 4 days.After that time, the cells were freeze-thawed three times. The celllysate was then titrated in CEF to select the recombinant viruses. After2 hrs of adsorption the viral inoculum was removed and a layer ofagarose containing EMEN was added. This layer was prepared by mixingidentical volumes of 2% low melting agarose and EMEN 2×. (Gibco, GrandIsland, N.Y.) with 4% fetal calf serum (Gibco, Grand Island, N.Y.). Atday four viral plaques were evident. CEFs transfected with pEFL-cr3 werestained by adding another agarose layer with 0.33% Xgal (MelfordLaboratories, UK) to the cultures. Blue plaques were selected andpurified three times until 100% of viral plaques were positive for Bgalactosidase expression. Stocks of lacZ+ viruses were then amplified inCEF grown in 25 cm2 flasks in 199 medium containing 10% tryptosephosphate broth plus 2% NBCS. The selected recombinant FPV was namedFPCR3.

[0057] Selective medium for transfection with plasmid pFP67xgptctlcontained mycophenolic acid (25 μg/ml), xantine (250 μg/ml) andhypoxantine (1 μg/ml). At day four viral plaques were evident. Since gptand cr3 genes are flanked by the same homology regions the isolation ofviral plaques in selective medium indicate that recombination occurredand both genes are inserted in FPV genome. Plaques were purified threeconsecutive times in CEF. The recombinant virus selected was namedFPSCR3GPT.

Example 5 PCR Analysis of FPCR3

[0058] PCR analysis was used to check that the FPCR3 recombinantscontained the cr3 gene. Recombinant FPV were propagated in CEFs for 6days and then the cells were harvested and pelleted. The pellet wassuspended and incubated for 2 h at 55° C. in 200 μl of extraction buffer(10 mM Tris HCl, 100 mM NaCl, 10 mM EDTA, 0.5% SDS, 2%β-mercaptoethanol) containing 1.25 mg/ml of proteinase K. The DNA wasthen phenol-chloroform extracted and ethanol precipitated. DNA from eachvirus was tested by PCR with the primers described below, complementaryto sequences in the 5′ and 3′ of cr3 gene, respectively. The PCRconditions used were 5 min at 94° C., followed by 25 cycles of 1 min at94° C., 1 min 30 sec at 45° C. and 1 min 30 sec at 72° C., and a finalextension at 72° C. for 10 min. The primer sequences were as follows:primer 775, 5′ TATTAACATTGCCTAGTAG 3′ primer 776,5′ GAAGTAGAATCATAAAGAAC 3′

[0059] Three independent CR3 recombinant viruses (FPCR3.1; FPCR3.2;FPCR3.3), showed the expected 1.3 kb band after the PCR reaction. Thisband was absent for the parental virus FPL29 (FIG. 3A).

Example 6 PCR Analysis FPSCR3GPT

[0060] PCR analysis was used to check that the FPSCR3GPT recombinantscontained the cr3 gene. Recombinant FPV were propagated in CEFs for 6days, and then the cells were harvested and pelleted. The pellet wassuspended and incubated for 2 h at 55° C. in 200 μl of extraction buffer(10 mM Tris HCl, 100 mM NaCl, 10 mM EDTA, 0.5% SDS, 2%α-mercaptoethanol) containing 1.25 mg/ml of proteinase K. The DNA wasthen phenol-chloroform extracted and ethanol precipitated. DNA from eachvirus was tested by PCR with the primers described below, complementaryto sequences in the 5′ and 3′ of cr3 gene, respectively. The PCRconditions used were 5 min at 94° C., followed by 25 cycles of 1 min at94° C., 1 min 30 sec at 45° C. and 1 min 30 sec at 72° C., and a finalextension at 72° C. for 10 min. The primer sequences were as follows:primer 2660, 5′ GAAGATCTGTACAGAAATGGAAAAG 3′  (257-279) primer 2663,5′ CCCTGCATGTGGCTCAACTGGTACTAGCTTG 3′ (1029-1059)

[0061] Three independent recombinant viruses (FPSCR3gpt.1; FPSCR3gpt.2;FPSCR3gpt.3), showed the expected 800 pb band after the PCR reaction.This band was absence for the parental virus FPL29 (FIG. 4A).

Example 7 Evaluation of CR3 Expression by CEF Infected by FPCR3

[0062] Expression of CR3 by the FPCR3 was confirmed by Western blotting.Confluent CEFs in 60 mm Petri dishes were infected at 0.5 pfu/cell withrecombinant FPV. After 24 hours the cells were harvested, pelleted andsuspended in 1×SDS gel-loading buffer (50 mM Tris HCl pH 6.8, 100 mMDTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol). Proteins werefractionated by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) on a 15% gel. They were thenelectro-transferred onto a nitrocellulose membrane (Hybond-C, Amersham,UK) following standard protocols. After transfer, the membrane wasblocked overnight in 5% non-fat dry milk in phosphate buffered saline(PBS: 2.68 mM KCl, 1.47 mM KH2PO4, 0.137M NaCl, 8.06 mM Na2HPO4). It wasthen incubated for 2 h at room temperature with 10 ug/ml of monoclonalantibody 6.2, diluted in PBS containing 1% dried milk. This monoclonalantibody was produced in mice immunized with CR3 (Iglesias E, Ruiz M,Carrazana Y, Cruz L J, Aguilar A, Jiménez V, Carpio E, Martfnez M, PerezM. Martinez C, Cruz O, Martin A, Duarte C. Chimeric proteins containingHIV-1 epitopes. Journal Biochemistry, Molecular Biology and Biophysics,2001, 5: 109-20.). The membrane was then washed and incubated with asheep anti-mouse antibody (1:2000) conjugated to horseradish peroxidase(HRPO) (Amersham, UK). After several washes, the immunoblots weredeveloped using the ECL Western blot detection system (Amersham, UK)according to the manufacturers' instructions. A specific band with amolecular weight between 50 y 64 kDa was detected in FPCR3 infectedcultures. No protein was detected in CEF infected with the parental FP9virus (FIG. 3B)

Example 8 Evaluation of CR3 Expression by CEF Infected by FPSCR3gpt

[0063] Expression of CR3 by the FPSCR3gpt was confirmed by Westernblotting following a procedure similar to the one described in theprevious example. A specific band with a molecular weight between 50 y64 kDa was also detected in FPSCR3gpt infected cultures while no proteinwas detected in CEF infected with the parental FP9 virus (FIG. 4B).

Example 9 Purification of FPCR3 and FPSCR3gpt and Immunization of Mice

[0064] Large stocks of recombinant FPV were grown on CEFs obtained fromeggs of a specific pathogen-free flock. FPV was purified bycentrifugation of cytoplasmic extracts through a 25% (w/v) sucrosecushion in a Beckman SW28 rotor at 29000 rpm for 2 hours. Virus titerswere then determined by plaque assay on CEF monolayers. FIG. 5 showsthat CR3 expression did not varies after scaling up of the culture.

[0065] Young adult (five to eight-week-old) female Balb/c mice (obtainedfrom the SPF breeding colony at the Institute for Animal Health,Compton, UK, or the Centro Nacional de Producción de Animales deLaboratorio (CENPALAB), Cuba) were primed by the intravenous (i.v),intraperitoneal (i.p), or subcutaneous (s.c) routes with 2.5-5×10⁷ pfuof FPCR3, FPSCR3gpt or the negative control virus in 200 μl sterile PBS.Two to four weeks later, mice were boosted by the same route with asecond dose of 2.5-9×10⁷ pfu of the same viruses in 200 μl sterile PBS.

Example 10 Detection of CTL Response Against CR3 in Balb/c Mice

[0066] Enzyme-linked-immunospot (ELISPOT) assays for detection ofantigen-specific IFN-γ-releasing cells were performed using a methodbased on that previously described (Tanguay S and J J Killion. Directcomparison of ELISPOT and ELISA-based assays for detection of individualcytokine-secreting cells. 1994. Lymphokine Cytokine Res, 13: 259-263).Briefly, immobilon-P membrane 96-well plates (Millipore, Molsheim,France) were coated with 100 μl/well of 5 μg/ml murine IFN-γ specificmonoclonal antibody R4 (Pharmingen, San Diego, Calif.) overnight at 4°C., washed 3× with PBS and blocked using RPMI 1640 medium supplementedwith 10% FBS at 37° C. for 1 h. Test cells were then added: these wereeither ex vivo splenocyte suspensions (prepared as described above) frommice primed and boosted with FPCR3 or FPSCR3gpt. Different numbers oftest cells were added per well: 10⁶, 2×10⁵ and 4×10⁴. Cells werestimulated by addition of P815 cells incubated with synthetic peptidesat 1 μM or infected with VV recombinant for CR3, Gag, or Nef at a m.o.iof 5 pfu/cell. P815 cells without peptide or infected with controlvaccinia viruses (vSC8 or wild type vaccinia strain WR) were included toreveal background numbers of IFN-γ-producing cells. Each well had afinal volume of 200 μl of R10 medium plus hIL-2. All assay variableswere tested in duplicate. After incubation overnight (at least 17hours), the plates were washed 3× with PBS and 5× with PBS plus 0.05%Tween 20, then a secondary biotin-conjugated antibody XMG1.2(Pharmingen, San Diego, Calif.) was added at 0.5 μg/ml and reacted atroom temperature for 2 h. The wells were washed 5× with PBS plus 0.05%Tween 20, and alkaline phosphatase (AP)-labeled streptavidin (VectorLabs, CA, USA) was added at a {fraction (1/1000)} dilution in PBS plus0.05% Tween 20 for 1 h at room temperature. The wells were washed again3× with PBS plus 0.05% Tween 20 and 3× with PBS, and the spots weredeveloped using an AP activity kit (Biorad, CA, USA). After 15 min, thewells were washed with tap water, dried and the spots counted under astereoscopic microscope (Leica Microscopy System, Heerbrugg,Switzerland). Alternatively, in some assays we used HRPO-labelledstreptavidin (Amersham, UK), diluted {fraction (1/800)}; spots were thendeveloped with 0.1% of 3,3′-diaminobenzidine (Sigma, Saint Louis, USA)in Tris-HCl 50 mM, pH 7.4 and 0.1% of hydrogen peroxide. The resultswere expressed as the number of spot-forming-cells (SFC) per 10⁶splenocytes or fractionated cells. Values more than twice the negativecontrol for each group (P815 without peptide or infected with controlVV) were considered positive.

[0067] Results from two independent ELISPOT assay are shown in FIG. 6. Asignificant fraction of splenocytes from Balb/c mice immunized withFPCR3 but no with negative virus was positive in IFN gamma ELISPOTagainst P815 infected either with VVCR3 or VVgag and VVnef or primedwith the V3 MN peptides (LKKKRIHIGPGRAFYERY).

[0068] In another experiment Balb/c mice were immunized with FPCR3 orFPSCR3gpt as described and the induction of CTLs was measured using aP815 stably transfected with cr3 (P815cr3). The results from thisexperiment are show in FIG. 7. Both recombinant FPV induced asignificant fraction of IFB gamma secreting cells specific for CR3.

Example 11 Proccesing and Recognition of CR3 Epitopes by LymphocytesFrom AIDS Patients

[0069] Autologous B cells from HIV infected patients were EBVtransformed and infected with a VV recombinant for CR3 (VVCR3). Thosetargets cells were incubated with peripheral blood lymphocytes from HIVpatients and the number of IFNγ secreting splenocytes were calculated byELISPOT. This experiment demonstrated that cr3 gene expressed bypoxvirus is capable to present efficiently its epitopes to CTLlymphocytes from HIV infected patients.

1 96 1 1333 DNA Human immunodeficiency virus type 1 1 atgcgtatcaaacagattat caacatgtgg caggaagtgg gcaaagcgat gtatgccccg 60 ccgatttctggtatggttga gcagatgcat gaagatatca ttagcctgtg ggaccagtct 120 cttaagaaaaagcgtatcca cattggccca ggccgtgcat tctatgaaag atacctaaag 180 gatcaacagctcctagggaa aaagcaactg ctgtttattc atttcagaat tgggtgtcga 240 catagcagaaagaaagagat ctgtacagaa atggaaaagg aagggaaaat ttcaaaaatt 300 gggcctgaaaatccatacaa tactccagta tttgctataa agaaaaaaga cagtactaaa 360 tggagaaaactagtagattt cagagaactt aataaaagaa ctcaagactt ctgggaagtt 420 cagttaggaataccacaccc cgcagggtta aaaaagaaaa aatcagtaac agtattggat 480 gtgggtgatgcatacttttc agttccctta gataaagact ttagaaagta tactgcattt 540 accatacctagtataaacaa tgagacacca gggattagat atcagtacaa tgtgctgcca 600 cagggatggaaaggatcacc agcaatattc caaagtagca tgacaaaaat cttagagcct 660 tttagaaaacagaatccaga catagttatc tatcaataca tggatgattt gtatgtagga 720 tcggacatcacaagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca 780 caagaggaggaggagatggg ttttccagtc acacctcagg tacctttaag accaatgact 840 tacaaggcagctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta 900 attcactcccaacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac 960 ttccctgattggcagaacta cacaccaggg ccaggggtca gatatccact gacctttgga 1020 tggtgctacaagctagtacc agttgagcca catgcagggc ctattgcacc aggccaaatg 1080 agagaaccaaggggaagtga catagcagga actactagta cccttcagga acaaatagga 1140 tggatgacaaataatccacc tatcccagta ggagaaatct ataaaagatg gataatcctg 1200 ggattaaataaaatagtaag aatgtatagc cctaccagct ttctggacat aagacaagga 1260 ccaaaggaaccctttagaga ttatgtagac cggttctata aaactctaag agccgaataa 1320 tctagaacggatc 1333 2 25 DNA Human immunodeficiency virus type 1 2 gaagatctgtacagaaatgg aaaag 25 3 31 DNA Human immunodeficiency virus type 1 3ggaattctcg cgatcctaca tacaaatcat c 31 4 23 DNA Human immunodeficiencyvirus type 1 4 gacatcacaa gtagcaatac agc 23 5 31 DNA Humanimmunodeficiency virus type 1 5 ccctgcatgt ggctcaactg gtactagctt g 31 628 DNA Human immunodeficiency virus type 1 6 gttgagccac atgcagggcctattgcac 28 7 33 DNA Human immunodeficiency virus type 1 7 gctctagattattcggctct tagagtttta tag 33 8 25 DNA Human immunodeficiency virus type1 8 gctctagatt attcggctct tagag 25 9 19 DNA Human immunodeficiency virustype 1 9 tattaacatt gcctagtag 19 10 20 DNA Human immunodeficiency virustype 1 10 gaagtagaat cataaagaac 20 11 25 DNA Human immunodeficiencyvirus type 1 11 gaagatctgt acagaaatgg aaaag 25 12 31 DNA Humanimmunodeficiency virus type 1 12 ccctgcatgt ggctcaactg gtactagctt g 3113 18 PRT Human immunodeficiency virus type 1 13 Leu Lys Lys Lys Arg IleHis Ile Gly Pro Gly Arg Ala Phe Tyr Glu 1 5 10 15 Arg Tyr 14 15 PRTHuman immunodeficiency virus type 1 14 His Ala Gly Pro Ile Ala Pro GlyGln Met Arg Glu Pro Arg Gly 1 5 10 15 15 20 PRT Human immunodeficiencyvirus type 1 15 Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp IleAla Gly 1 5 10 15 Thr Thr Ser Thr 20 16 20 PRT Human immunodeficiencyvirus type 1 16 Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu GlnIle Gly 1 5 10 15 Trp Met Thr Asn 20 17 10 PRT Human immunodeficiencyvirus type 1 17 Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp 1 5 10 18 15 PRTHuman immunodeficiency virus type 1 18 Asn Pro Pro Ile Pro Val Gly GluIle Tyr Lys Arg Trp Ile Ile 1 5 10 15 19 22 PRT Human immunodeficiencyvirus type 1 19 Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp IleIle Leu 1 5 10 15 Gly Leu Asn Lys Ile Val 20 20 9 PRT Humanimmunodeficiency virus type 1 20 Pro Pro Ile Pro Val Gly Glu Ile Tyr 1 521 15 PRT Human immunodeficiency virus type 1 21 Ile Pro Val Gly Glu IleTyr Lys Arg Trp Ile Ile Leu Gly Leu 1 5 10 15 22 9 PRT Humanimmunodeficiency virus type 1 22 Gly Glu Ile Tyr Lys Arg Trp Ile Ile 1 523 9 PRT Human immunodeficiency virus type 1 23 Glu Ile Tyr Lys Arg TrpIle Ile Leu 1 5 24 10 PRT Human immunodeficiency virus type 1 24 Ile TyrLys Arg Trp Ile Ile Leu Gly Leu 1 5 10 25 19 PRT Human immunodeficiencyvirus type 1 25 Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Thr Val ArgMet Tyr 1 5 10 15 Ser Pro Thr 26 9 PRT Human immunodeficiency virus type1 26 Lys Arg Trp Ile Ile Leu Gly Leu Asn 1 5 27 10 PRT Humanimmunodeficiency virus type 1 27 Ile Ile Leu Gly Leu Asn Lys Ile Val Arg1 5 10 28 10 PRT Human immunodeficiency virus type 1 28 Leu Gly Leu AsnLys Ile Val Arg Met Tyr 1 5 10 29 11 PRT Human immunodeficiency virustype 1 29 Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser 1 5 10 30 9 PRTHuman immunodeficiency virus type 1 30 Gly Leu Asn Lys Ile Val Arg MetTyr 1 5 31 19 PRT Human immunodeficiency virus type 1 31 Leu Asp Ile ArgGln Gly Pro Lys Glu Pro Arg Asp Tyr Val Asp Arg 1 5 10 15 Phe Tyr Lys 3211 PRT Human immunodeficiency virus type 1 32 Arg Asp Tyr Val Asp ArgPhe Tyr Lys Thr Leu 1 5 10 33 9 PRT Human immunodeficiency virus type 133 Asp Arg Phe Tyr Lys Thr Leu Arg Ala 1 5 34 9 PRT Humanimmunodeficiency virus type 1 34 Phe Pro Val Thr Pro Gln Val Pro Leu 1 535 10 PRT Human immunodeficiency virus type 1 35 Phe Pro Val Thr Pro GlnVal Pro Leu Arg 1 5 10 36 9 PRT Human immunodeficiency virus type 1 36Thr Pro Gln Val Pro Leu Arg Pro Met 1 5 37 8 PRT Human immunodeficiencyvirus type 1 37 Val Pro Leu Arg Pro Met Thr Tyr 1 5 38 10 PRT Humanimmunodeficiency virus type 1 38 Gln Val Pro Leu Arg Pro Met Thr Tyr Lys1 5 10 39 8 PRT Human immunodeficiency virus type 1 39 Val Pro Leu ArgPro Met Thr Tyr 1 5 40 8 PRT Human immunodeficiency virus type 1 40 ProLeu Arg Pro Met Thr Tyr Lys 1 5 41 10 PRT Human immunodeficiency virustype 1 41 Lys Ala Ala Val Asp Leu Ser His Phe Leu 1 5 10 42 12 PRT Humanimmunodeficiency virus type 1 42 Ala Ala Val Asp Leu Ser His Phe Leu LysGlu Lys 1 5 10 43 8 PRT Human immunodeficiency virus type 1 43 Ala ValAsp Leu Ser His Phe Leu 1 5 44 9 PRT Human immunodeficiency virus type 144 Ala Val Asp Leu Ser His Phe Leu Lys 1 5 45 9 PRT Humanimmunodeficiency virus type 1 45 Asp Leu Ser His Phe Leu Lys Glu Lys 1 546 15 PRT Human immunodeficiency virus type 1 46 Asp Leu Ser His Phe LeuLys Glu Lys Gly Gly Leu Glu Gly Leu 1 5 10 15 47 8 PRT Humanimmunodeficiency virus type 1 47 Phe Leu Lys Glu Lys Gly Gly Leu 1 5 489 PRT Human immunodeficiency virus type 1 48 Lys Glu Lys Gly Gly Leu GluGly Leu 1 5 49 14 PRT Human immunodeficiency virus type 1 49 Glu Lys GlyGly Leu Glu Gly Leu Ile His Ser Gln Arg Arg 1 5 10 50 14 PRT Humanimmunodeficiency virus type 1 50 His Ser Gln Arg Arg Gln Asp Ile Leu AspLeu Trp Ile Tyr 1 5 10 51 25 PRT Human immunodeficiency virus type 1 51Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile Tyr His Thr Gln 1 5 1015 Gly Tyr Phe Pro Asp Trp Gln Asn Tyr 20 25 52 10 PRT Humanimmunodeficiency virus type 1 52 Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile1 5 10 53 10 PRT Human immunodeficiency virus type 1 53 Arg Gln Asp IleLeu Asp Leu Trp Ile Tyr 1 5 10 54 11 PRT Human immunodeficiency virustype 1 54 Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln 1 5 10 55 10 PRTHuman immunodeficiency virus type 1 55 His Thr Gln Gly Tyr Phe Pro AspTrp Gln 1 5 10 56 12 PRT Human immunodeficiency virus type 1 56 Thr GlnGly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr 1 5 10 57 11 PRT Humanimmunodeficiency virus type 1 57 Thr Gln Gly Tyr Phe Pro Asp Trp Gln AsnTyr 1 5 10 58 9 PRT Human immunodeficiency virus type 1 58 Tyr Phe ProAsp Trp Gln Asn Tyr Thr 1 5 59 25 PRT Human immunodeficiency virus type1 59 Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly Pro Gly Ile Arg Tyr 1 510 15 Pro Leu Thr Phe Gly Trp Cys Tyr Lys 20 25 60 13 PRT Humanimmunodeficiency virus type 1 60 Asn Tyr Thr Pro Gly Pro Gly Val Arg TyrPro Leu Thr 1 5 10 61 11 PRT Human immunodeficiency virus type 1 61 ThrPro Gly Pro Gly Val Arg Tyr Pro Leu Thr 1 5 10 62 14 PRT Humanimmunodeficiency virus type 1 62 Gly Pro Gly Val Arg Tyr Pro Leu Thr PheGly Trp Cys Tyr 1 5 10 63 16 PRT Human immunodeficiency virus type 1 63Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu Val Pro 1 5 1015 64 16 PRT Human immunodeficiency virus type 1 64 Val Arg Tyr Pro LeuThr Phe Gly Trp Cys Tyr Lys Leu Val Pro Val 1 5 10 15 65 9 PRT Humanimmunodeficiency virus type 1 65 Tyr Pro Leu Thr Phe Gly Trp Cys Tyr 1 566 10 PRT Human immunodeficiency virus type 1 66 Pro Leu Thr Phe Gly TrpCys Tyr Lys Leu 1 5 10 67 17 PRT Human immunodeficiency virus type 1 67Glu Ile Cys Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly 1 5 1015 Pro 68 9 PRT Human immunodeficiency virus type 1 68 Glu Lys Glu GlyLys Ile Ser Lys Ile 1 5 69 9 PRT Human immunodeficiency virus type 1 69Gly Ile Pro His Pro Ala Gly Leu Lys 1 5 70 16 PRT Human immunodeficiencyvirus type 1 70 Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp ValGly Asp 1 5 10 15 71 15 PRT Human immunodeficiency virus type 1 71 LysLys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser 1 5 10 15 72 9PRT Human immunodeficiency virus type 1 72 Thr Val Leu Asp Val Gly AspAla Tyr 1 5 73 11 PRT Human immunodeficiency virus type 1 73 Val Leu AspVal Gly Asp Ala Tyr Phe Ser Val 1 5 10 74 8 PRT Human immunodeficiencyvirus type 1 74 Asp Ala Tyr Phe Ser Val Pro Leu 1 5 75 10 PRT Humanimmunodeficiency virus type 1 75 Val Pro Leu Asp Glu Asp Phe Arg Lys Tyr1 5 10 76 10 PRT Human immunodeficiency virus type 1 76 Lys Tyr Thr AlaPhe Thr Ile Pro Ser Ile 1 5 10 77 8 PRT Human immunodeficiency virustype 1 77 Thr Ala Phe Thr Ile Pro Ser Ile 1 5 78 9 PRT Humanimmunodeficiency virus type 1 78 Gln Gly Trp Lys Gly Ser Pro Ala Ile 1 579 13 PRT Human immunodeficiency virus type 1 79 Trp Lys Gly Ser Pro AlaIle Phe Gln Ser Ser Met Thr 1 5 10 80 9 PRT Human immunodeficiency virustype 1 80 Ser Pro Ala Ile Phe Gln Ser Ser Met 1 5 81 9 PRT Humanimmunodeficiency virus type 1 81 Ala Ile Phe Gln Ser Ser Met Thr Lys 1 582 9 PRT Human immunodeficiency virus type 1 82 Lys Gln Asn Pro Asp IleVal Ile Tyr 1 5 83 9 PRT Human immunodeficiency virus type 1 83 Asn ProAsp Ile Val Ile Tyr Gln Tyr 1 5 84 9 PRT Human immunodeficiency virustype 1 84 Val Ile Tyr Gln Tyr Met Asp Asp Leu 1 5 85 11 PRT Humanimmunodeficiency virus type 1 85 Val Ile Tyr Gln Tyr Met Asp Asp Leu TyrVal 1 5 10 86 20 PRT Human immunodeficiency virus type 1 86 Phe Arg LysGln Asn Pro Asp Ile Val Ile Tyr Gln Tyr Met Asp Asp 1 5 10 15 Leu TyrVal Gly 20 87 20 PRT Human immunodeficiency virus type 1 87 Lys Gln IleIle Asn Met Trp Gln Glu Val Gly Lys Ala Met Tyr Ala 1 5 10 15 Pro ProIle Glu 20 88 7 PRT Human immunodeficiency virus type 1 88 Lys Val GlyLys Ala Met Tyr 1 5 89 8 PRT Human immunodeficiency virus type 1 89 LysVal Gly Lys Ala Met Tyr Ala 1 5 90 13 PRT Human immunodeficiency virustype 1 90 His Glu Asp Ile Ile Ser Leu Trp Asn Gln Ser Leu Lys 1 5 10 919 PRT Human immunodeficiency virus type 1 91 Ile Ile Ser Leu Trp Asn GlnSer Leu 1 5 92 11 PRT Human immunodeficiency virus type 1 92 Glu Arg TyrLeu Lys Asp Gln Gln Leu Leu Gly 1 5 10 93 8 PRT Human immunodeficiencyvirus type 1 93 Tyr Leu Lys Asp Gln Gln Leu Leu 1 5 94 10 PRT Humanimmunodeficiency virus type 1 94 Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu1 5 10 95 8 PRT Human immunodeficiency virus type 1 95 Arg Tyr Leu LysAsp Gln Gln Leu 1 5 96 15 PRT Human immunodeficiency virus type 1 96 GlnLeu Leu Phe Ile His Phe Arg Ile Gly Cys Arg His Ser Arg 1 5 10 15

1. A chimeric gene containing fragments from different HIV-1 genes,where those fragments encodes for cytotoxic T cells (CTL) epitopes richregions, which are presented by a wide range of Major HistocompatiblityComplex (HLA-1) antigens, and can also contain T helper (Th) cellsepitopes from HIV and at least one B cell epitope that is the target ofa monoclonal antibody.
 2. A gene as described in claim 1 which encodesfor a chimeric poliprotein containing fragments from at least one HIVstructural protein and one HIV non-structural protein.
 3. A gene asdescribed in claim 2 which encodes for a chimeric poliprotein containingfragments from HIV-1 proteins Reverse Transcriptase, P24 and Nef, Thepitopes from gp120, gp41 and vpr and a B cell epitope from gp120.
 4. Agene as described in claim 3 which encodes for a chimeric poliproteincontaining fragments 203-259 from Reverse Transcriptase, 219-307 fromP24, and 45-147 from Nef., Th cell epitopes T1 and T2 from gp120,580-594 from gp41 and 566-580 from vpr and B cell epitope from the V3region MN strain recognized by Mab 2C4.
 5. A gene as described in claim4, which DNA sequence corresponds essentially with that of cr3 gene. 6.A chimeric protein which amino acid sequence corresponds essentiallywith the sequence of the protein CR3.
 7. A recombinant virus for anheterologous gene, which contains fragments from different HIV-1 genes,where those fragments encodes for CTL epitopes rich regions, which arepresented by a wide range of HLA-1 antigens, and can contain also HIV-1T helper cell epitopes and at least one B cell epitope recognized by aMab.
 8. A recombinant virus as described in claim 7 where theheterologous gene encodes for a chimeric protein containing fragmentsfrom at least one HIV structural protein and one HIV non-structuralprotein.
 9. A recombinant virus as described in claim 8 where theheterologous gene encodes for a chimeric protein containing fragmentsfrom HIV-1 proteins RT, P24 and Nef, Th epitopes from gp120, gp41 andvpr and a B cell epitope from gp120.
 10. A recombinant virus asdescribed in claim 9 where the heterologous gene encodes for a chimericprotein containing fragments 203-259 from RT; 219-307 from P24, and45-147 from NEF and Th cell epitopes T1 and T2 from gp120, 580-594 fromgp41 and 566-580 from vpr and B cell epitope from the V3 region MNstrain recognized by Mab 2C4.
 11. A recombinant virus as described inclaim 10 where the DNA sequence of the heterologous gene correspondsessentially with cr3.
 12. A virus as described in claims 7-11 where thisvirus is a poxvirus.
 13. A virus as described in claims 7-12 where thisvirus is an Avipoxvirus
 14. A virus as described in claims 7-13 wherethis virus is Fowl Pox Virus.
 15. A virus as described in claims 7-14where this virus is FPCR3.
 16. A virus as described in claims 7-14 wherethis virus is FPSCR3gpt.
 17. A vaccine formulation containing: Arecombinant virus as described in claims 7-16. A pharmaceuticalacceptable vehicle.
 18. The use of a vaccine formulation described inclaim 17 to induce an immune response against HIV in AIDS patients oruninfected persons.
 19. A preventive or therapeutic combination composedof the vaccine formulation described in claim 17 and animmunopotentiator substance.
 20. A preventive or therapeutic combinationas described in claim 19 where immunopotentiator substance is acytokine.
 21. A preventive or therapeutic combination as described inclaim 20 where such cytokine is IL2.
 22. A plasmid verctor containingchimeric gene as described in claims 1-5 under the control of amammalian cells promoter.
 23. A vaccine formulation containing: Arecombinant plasmid vector as described in claim 22 A pharmaceuticalacceptable vehicle.
 24. The use of a vaccine formulation described inclaim 23 to induce an immune response against different proteins of HIVin AIDS patients or uninfected persons.
 25. A preventive or therapeuticcombination composed of the vaccine formulation described in claim 23and an immunopotentiator substance.
 26. A preventive or therapeuticcombination as described in claim 25 where immunopotentiator substanceis a cytokine.
 27. A preventive or therapeutic combination as describedin claim 26 where such cytokine is IL2.